Organ reconstruction

ABSTRACT

The invention is directed to methods and devices for the reconstruction, repair, augmentation or replacement of laminarily organized organs or tissue structures in a patient in need of such treatment. The device comprises a biocompatible synthetic or natural polymeric matrix shaped to conform to at least a part of the luminal organ or tissue structure with a first cell population on or in a first area and a second cell population such as a smooth muscle cell population in a second area of the polymeric-matrix. The method involves grafting the device to an area in a patient in need of treatment. The polymeric matrix comprise a biocompatible and biodegradable material.

BACKGROUND

[0001] 1. Field of the Invention

[0002] The invention is directed to methods and materials for tissuereconstruction, repair augmentation and replacement, and particularly touse of such treatments in patients having a defect in urogenital tissuessuch as the bladder.

[0003] 2. Description of the Background

[0004] The medical community has directed considerable attention andeffort to the substitution of defective organs with operationallyeffective replacements. The replacements have ranged from completelysynthetic devices such as artificial hearts to completely natural organsfrom another mammalian donor. The field of heart transplants has beenespecially successfully with the use of both synthetic hearts to naturalhearts from living donors. Equal success has not been achieved in manyother organ fields particularly in the field of bladder reconstruction.

[0005] The human urinary bladder is a musculomembranous sac, situated inthe anterior part of the pelvic cavity, that serves as a reservoir forurine, which it receives through the ureters and discharges through theurethra. In a human the bladder is found in the pelvis behind the pelvicbone (pubic symphysis) and a drainage tube, called the urethra, thatexits to the outside of the body. The bladder, ureters, and urethra areall similarly structured in that they comprise muscular structures linedwith a membrane comprising urothelial cells coated with mucus that isimpermeable to the normal soluble substances of the urine. The trigoneof the bladder, also called the trigonum vesicae, is a smooth triangularportion of the mucous membrane at the base of the bladder. The bladdertissue is elastic and compliant. That is, the bladder changes shape andsize according to the amount of urine it contains. A bladder resembles adeflated balloon when empty but becomes somewhat pear-shaped and risesinto the abdominal cavity when the amount of urine increases.

[0006] The bladder wall has three main layers of tissues: the mucosa,submucosa, and detrusor. The mucosa, comprising urothelial cells, is theinnermost layer and is composed of transitional cell epithelium. Thesubmucosa lies immediately beneath the mucosa and its basement membrane.It is composed of blood vessels which supply the mucosa with nutrientsand the lymph nodes which aid in the removal of waste products. Thedetrusor is a layer of smooth muscle cells which expands to store urineand contracts to expel urine.

[0007] The bladder is subjected to numerous maladies and injuries whichcause deterioration in patients. For example, bladder deterioration mayresult from infectious diseases, neoplasms and developmentalabnormalities. Further, bladder deterioration may also occur as a resultof trauma such as, for example, car accidents and sports injury.

[0008] Although a large number of bio-materials, including synthetic andnaturally-derived polymers, have been employed for tissue reconstructionor augmentation (see, e.g., “Textbook or Tissue Engineering” Eds. Lanza,R., Langer, R, and Chick, W, ACM Press, Colorado (1996) and referencescited therein), no material has proven satisfactory for use in bladderreconstruction. For example, synthetic biomaterials such as polyvinyland gelatin sponges, polytetrafluoroethylene (Teflon) felt, and silasticpatches have been relatively unsuccessful, generally due to foreign bodyreactions (see, e.g., Kudish, H. G., J. Urol. 78:232 (1957); Ashkar, L.and Heller, E., J. Urol. 98:91 (1967); Kelami, A. et al., J. Urol.104:693 (1970)). Other attempts have usually failed due to eithermechanical, structural, functional, or biocompatibility problems.Permanent synthetic materials have been associated with mechanicalfailure and calculus formation.

[0009] Naturally-derived materials such as lyophilized dura,de-epithelialized bowel segments, and small intestinal submucosa (SIS)have also been proposed for bladder replacement (for a general review,see Mooney, D. et al., “Tissue Engineering: Urogenital System” in“Textbook of Tissue Engineering” Eds. Lanza, R., Langer, R, and Chick,W., ACM Press, Colorado (1996)). However, it has been reported thatbladders augmented with dura, peritoneum, placenta and fascia contractover time (Kelami, A. et al., J. Urol. 105:518 (1971)). De-epithelizedbowel segments demonstrated an adequate urothelial covering for use inbladder reconstruction, but difficulties remain with either mucosalregrowth, segment fibrosis, or both. It has been shown thatde-epithelization of the intestinal segments may lead to mucosalregrowth whereas removal of the mucosa and submucosa may lead toretraction of the intestinal segment (see, e.g., Atala, A., J. Urol.156:338 (1996)).

[0010] Other problems have been reported with the use of certaingastrointestinal segments for bladder surgery including stone formation,increased mucus production, neoplasia, infection, metabolicdisturbances, long term contracture and resorption. These attempts withnatural or synthetic materials have shown that bladder tissue, with itsspecific muscular elastic properties and urothelial permeabilityfunctions, cannot be easily replaced.

[0011] Due to the multiple complications associated with the use ofgastrointestinal segments for bladder reconstruction, investigators havesought alternate solutions. Recent surgical approaches have relied onnative urological tissue for reconstruction, including auto-augmentationand ureterocystoplasty. However, autoaugmentation has been associatedwith disappointing long-term results and ureterocystoplasty is limitedto cases in which a dilated ureter is already present. A system ofprogressive dilation for ureters and bladders has been proposed,however, this has not yet been attempted clinically. Sero-musculargrafts and de-epithelialized bowel segments, either alone or over anative urothelium, have also been attempted. However, graft shrinkageand re-epithelialization of initially de-epithelialized bowel segmentshas been a recurring problem.

[0012] One significant limitation besetting bladder reconstruction isdirectly related to the availability of donor tissue. The limitedavailability of bladder tissue prohibits the frequent routinereconstruction of bladder using normal bladder tissue. The bladdertissue that is available, and considered usable, may itself includeinherent imperfections and disease. For example, in a patient sufferingfrom bladder cancer, the remaining bladder tissue may be contaminatedwith metastasis. Accordingly, the patient is predestined to less thanperfect bladder function.

SUMMARY OF THE INVENTION

[0013] The present invention overcomes the problems and disadvantagesassociated with current strategies for reconstruction repair ofaugmentation and replacement of luminal organs and tissue structures.

[0014] One embodiment of this invention is directed to a method for thereconstruction, repair, augmentation or replacement of laminarilyorganized luminal organs or tissue structures in a patient in need ofsuch treatment. The method involves providing a biocompatible syntheticor natural polymeric matrix shaped to conform to at least a part of theluminal organ or tissue structure in need of said treatment, depositinga first cell population on or in a first area of said polymeric matrix,depositing a second cell population of a different cell type than saidfirst cell population in a second area of the polymeric matrix. Thesecond area is substantially separated from the first area. The shapedpolymeric matrix cell construct is implanted into the patient at thesite in need of treatment to form a laminarily organized luminal organor tissue structure. Another embodiment of this invention is directed toa device for the reconstruction, repair, augmentation or replacement ofluminarily organized luminal organs or tissue structures. The devicecomprises an implantable, biocompatible, synthetic or natural polymericmatrix with at least two separate surfaces. The polymeric matrix isshaped to conform to a at least a part of the luminal organ or tissuestructure in need of said treatment and at least two different cellpopulations are deposited in substantially separate areas either on orin the polymeric matrix to form a luminarily organized matrix/cellconstruct.

[0015] A further embodiment of this invention is directed to a devicefor the repair, reconstruction, augmentation or replacement of damagedor missing bladder tissue in a patient in need of such treatment. Thedevice comprises an implantable, biocompatible synthetic or naturalpolymeric matrix which is shaped to conform to the part of a bladdertissue in need of treatment. Urothelial cells are deposited on and nearthe inside surface of the matrix, and smooth muscle cells are depositedon and near the outside surface of said matrix. Upon implantation intothe patient, the device forms a laminarily organized luminal tissuestructure with the compliance of normal bladder tissue.

[0016] Other embodiments and advantages of the invention are set forth,in part, in the description which follows and, in part, will be obviousfrom this description and may be learned from the practice of theinvention.

DESCRIPTION OF THE DRAWINGS

[0017] The rule of this patent contains at least one drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee,

[0018]FIG. 1 depicts (A) a native canine bladder prior totrigone-sparing cystectomy; (B) an engineered neo-Organ anastomosed tothe trigone; and (C) an implant, decompressed by a transurethral andsuprapubic catheter, wrapped with omentum.

[0019]FIG. 2 depicts (A) bladder capacities and (B) compliance atdifferent postoperative time points relative to preoperative capacitiesof 100%.

[0020]FIG. 3 depicts radiographic cystograms 11 months after subtotalcystectomy followed by (A) Subtotal cystectomy without reconstruction(Group A); (B) Polymer only implant (Group B); and (C) tissue engineeredneo-organ (Group C).

[0021]FIG. 4 depicts (A and B) gross aspect of subtotal cystectomycontrol; (C and D) polymer only implant; and (E and F) tissue engineeredneo-organ retrieved after 11 months.

[0022]FIG. 5 depicts H&E histological results six months after surgeryof (A) normal canine bladder; (B) bladder dome of the cell-free polymerreconstructed bladder (group B); (C) the tissue engineered neoorgan(group C).

[0023]FIG. 6 depicts positive immunocytochemical staining of tissueengineered neo-organ for (A) pancytokeratins AE1/AE3; (B) Urothelialdifferentiation related membrane proteins; (C) smooth muscle actin; and(D) S-100 antibodies six months after implantation.

DESCRIPTION OF THE INVENTION

[0024] The present invention provides methods and devices thatfacilitate tissue reconstruction. In its broadest form, the methods anddevices of the present invention are useful in the reconstruction,repair, augmentation or replacement of organs or tissues structures thatcomprise multilayer cellular organization and particularly those organsor tissue structures that are luminal in nature. More particularly, thepresent invention provides methods and devices that facilitate thereconstruction, repair, augmentation or replacement of shaped holloworgans or tissue structures that exhibit a laminar segregation ofdifferent cell types and that have a need to retain a general luminalshape. Luminal organs or tissue structures that contain a smooth musclecell (SMC) layer to impart compliant or contractible properties to theorgan or structure are particularly well suited to the methods anddevices of the present invention.

[0025] In an example of one preferred embodiment of the invention, theluminal organ is the bladder, which has an inner layer of a first cellpopulation that comprises urothelial cells and an outer layer of asecond cell population that comprises smooth muscle cells. Thisorganization is also present in other genitourinary organs and tissuestructures such as the ureters and urethra. Laminarily organized organsor tissues refer to any organ or tissue made up of; or arranged inlaminae including ductal tissue. Other suitable laminarily organizedluminal organs, tissue structure, or ductal tissues to which the presentinvention is directed include vas deferens, fallopian tubes, lacrimalducts, trachea, stomach, intestines, vasculature, biliary duct, ductusejaculatorius, ductus epididymidis, ductus parotideus, and surgicallycreated shunts.

[0026] The method of the present invention in its broadest aspectencompasses as a first step providing a biocompatible synthetic ornatural polymeric matrix that is shaped to conform to its use as a partor all of the luminal organ or tissue structure to be repaired,reconstructed, augmented or replaced. A biocompatible material is anysubstance not having toxic or injurious effects on biological function.The shaped matrix is preferably porous to allow for cell deposition bothon and in the pores of the matrix. The shaped polymeric matrix is thencontacted, preferably sequentially, With at least two different cellpopulations supplied to separate areas of the matrix (e.g., inside andoutside) to seed the cell population on and/or into the matrix. Theseeded matrix is then implanted in the body of the recipient where theseparate, laminarily organized cell populations facilitate the formationof neo-organs or tissue structures.

[0027] In a preferred embodiment, the materials and methods of theinvention are useful for the reconstruction or augmentation of bladdertissue. Thus, the invention provides treatments for such conditions asbladder exstrophy, bladder volume insufficiency, reconstruction ofbladder following partial or total cystectomy, repair of bladdersdamaged by trauma, and the like.

[0028] While reference is made herein to augmentation of bladderaccording to the invention, it will be understood that the methods andmaterials of the invention are useful for tissue reconstruction oraugmentation of a variety of tissues and organs in a subject. Thus, forexample, organs or tissues such as bladder, ureter, urethra, renalpelvis, and the like, can be augmented or repaired with polymericmatrixes seeded with cells. The materials and methods of the inventionfurther can be applied to the reconstruction or augmentation of vasculartissue (see, e.g., Zdrahala, R. J., J Biomater. Appl. 10 (4): 309-29(1996)), intestinal tissues, stomach (see, e.g., Laurencin, C.T. et al.,J Biomed Mater. Res. 30 (2): 133-8 1996), and the like. The patient tobe treated may be of any species of mammals such as a dog, cat, pig,horse, cow, or human, in need of reconstruction, repair, or augmentationof a tissue. Polymeric matrices Biocompatible material and especiallybiodegradable material is the preferred material for the construction ofthe polymeric matrix. The polymeric matrix is used in the constructionof the reconstructive urothelial graft (RUG). The RUG is an implantable,biocompatible, synthetic or natural polymeric matrix with at least twoseparate surfaces. The RUG is shaped to conform to a at least a part ofthe luminal organ or tissue structure in need or treatment and has atleast two different cell populations deposited in substantially separateareas either on or in the polymeric matrix. Thus the RUG is a laminarilyorganized matrix/cell construct.

[0029] Biocompatible refers to materials which do not have toxic orinjurious effects on biological functions. Biodegradable refers tomaterial that can be absorbed or degraded in a patient's body. Examplesof biodegradable materials include, for example, absorbable sutures.Representative materials for forming the biodegradable structure includenatural or synthetic polymers, such as, for example, collagen,poly(alpha esters) such as poly(lactate acid), poly(glycolic acid),polyorthoesters and polyanhydrides and their copolymers, which degradedby hydrolysis at a controlled rate and are reabsorbed. These materialsprovide the maximum control of degradability, manageability, size andconfiguration. Preferred biodegradable polymer material includepolyglycolic acid and polyglactin, developed as absorbable syntheticsuture material. Polyglycolic acid and polyglactin fibers may be used assupplied by the manufacturer. Other biodegradable materials includecellulose ether, cellulose, cellulosic ester, fluorinated polyethylene,poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,polyester, polyestercarbonate, polyether, polyetheretherketone,polyetherimide, polyetherketone, polyethersulfone, polyethylene,polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenyleneoxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, or copolymers or physical blends of thesematerials. The material may be impregnated with suitable antimicrobialagents and may be colored by a color additive to improve visibility andto aid in surgical procedures.

[0030] A presently preferred biocompatible polymer is Polyglactin,developed as absorbable synthetic suture material, a 90:10 copolymer ofglycolide and lactide, manufactured as Vicryl braided absorbable suture(Ethicon Co., Somerville, N.J.) (Craig P. H., Williams J. A., Davis K.W., et al.: A Biological Comparison of Polyglactin 910 and PolyglycolicAcid Synthetic Absorbable Sutures. Surg. 141; 1010, (1975)) andpolyglycolic acid. Polyglactin and polyglycolic acid fibers can be usedas supplied by the manufacturer. The biocompatible polymer may be shapedusing methods such as, for example, solvent casting, compressionmolding, filament drawing, meshing, leaching, weaving and coating. Insolvent casting, a solution of one or more polymers in an appropriatesolvent, such as methylene chloride, is cast as a branching patternrelief structure. After solvent evaporation, a thin film is obtained. Incompression molding, a polymer is pressed at pressures up to 30,000pounds per square inch into an appropriate pattern. Filament drawinginvolves drawing from the molten polymer and meshing involves forming amesh by compressing fibers into a felt-like material. In leaching, asolution containing two materials is spread into a shape close to thefinal form of the RUG. Next a solvent is used to dissolve away one ofthe components, resulting in pore formation. (See Mikos, U.S. Pat. No.5,514,378, hereby incorporated by reference). In nucleation, thin filmsin the shape of a RUG are exposed to radioactive fission products thatcreate tracks of radiation damaged material. polycarbonate sheets areetched with acid or base, turning the tracks of radiation damagedmaterial into pores. Finally, a laser may be used to shape and burnindividual holes through many materials to form a RUG structure withuniform pore sizes. Coating refers to coating or permeating a polymericstructure with a material such as, for example liquefied copolymers(poly-DL-lactide co-glycolide 50:50 80 mg/ml methylene chloride) toalter its mechanical properties. Coating may be performed in one layer,or multiple layers until the desired mechanical properties are achieved.These shaping techniques may be employed in combination, for example, apolymeric matrix may be weaved, compression molded and glued together.Furthermore different polymeric materials shaped by different processesmay be joined together to form a composite shape. The composite shapemay be a laminar structure. For example, a polymeric matrix may beattached to one or more polymeric matrixes to form a multilayerpolymeric matrix structure. The attachment may be performed by gluingwith a liquid polymer or by suturing. In addition, the polymeric matrixmay be formed as a solid block and shaped by laser or other Standardmachining techniques to its desired final form. Laser shaping refers tothe process of removing materials using a laser.

[0031] The biodegradable polymers can be characterized with respect tomechanical properties, such as tensile strength using an Instron tester,for polymer molecular weight by gel permeation chromatography (GPC),glass, transition temperature by differential scanning calorimetry (DSC)and bond structure by infrared (IR) spectroscopy; with respect totoxicology by initial screening tests involving Ames assays and in vitroteratogenicity assays and implantation studies in animals forimmunogenicity, inflammation, release and degradation studies. In vitrocell attachment and viability can be assessed using scanning electronmicroscopy, histology and quantitative assessment with radioisotopes.The biodegradable material may also be characterized with respect to theamount of time necessary for the material to degrade when implanted in apatient. By varying the construction, such as, for example, thethickness and mesh size, the biodegradable material may substantiallybiodegrade between about 2 years or about 2 months, preferably betweenabout 18 months and about 4 months, most preferably between about 15months and about 8 months and most preferably between about 12 monthsand about 10 months. If necessary, the biodegradable material may beconstructed so as not to degrade substantially within about 3 years, orabout 4 years or about five or more years.

[0032] The polymeric matrix may be fabricated with controlled porestructure as described above. The size of the pores may be used todetermine the cell distribution. For example, the pores on the polymericmatrix may be large to enable cells to migrate from one surface to theopposite surface. Alternatively, the pores may be small such that thereis fluid communication between the two sides of the polymeric matrix butcells cannot pass through. Suitable pore size to accomplish thisobjective may be about 0.04 micron to about 10 microns in diameter,preferably between about 0.4 micron to about 4 microns in diameter. Insome embodiments, the surface of the polymeric matrix may comprise poressufficiently large to allow attachment and migration of a firstpopulation of cells into the pores. The pore size may be reduced in theinterior of the polymeric matrix to prevent cells from migrating fromone side of the polymeric matrix to the opposite side. On the oppositeside of the polymeric matrix, the pores may again enlarge to allow theattachment and establishment of a second population of cells. Because ofthe reduced pore size in the interior of the polymeric matrix, the firstcell population and the second cell population initially cannot mix. Oneembodiment of a polymeric matrix with reduced pore size is a laminatedstructure of a small pore material sandwiched between two large porematerial. Alternatively, a large pore material laminated to a small porematerial may also allow cells to establish growth on both sides withoutany intermixing of cells. Polycarbonate membranes are especiallysuitable because they can be fabricated in very controlled pore sizessuch as, for example, about 0.01 microns, about 0.05 micron, about 0.1micron, about 0.2 micron, about 0.45 micron, about 0.6 micron, about 1.0micron, about 2.0 microns and about 4.0 microns. At the submicron levelthe polymeric matrix may be impermeable to bacteria, viruses and othermicrobes. At the present time, a mesh-like structure formed of fibers,which may be round, scalloped, flattened, star shaped, solitary orentwined with other fibers is preferred. The use of branching fibers isbased upon the same principles which nature has used to solve theproblem of increasing surface area proportionate to volume increases.All multicellular organisms utilize this repeating branching structure.Branching systems represent communication networks between organs, aswell as the functional units of individual organs. Seeding andimplanting this configuration with cells allows implantation of largenumbers of cells, each of which is exposed to the environment of thehost, providing for free exchange of nutrients and waste whileneovascularization is achieved. The polymeric matrix may be madeflexible or rigid, depending on the desired final form, structure andfunction.

[0033] In one preferred embodiment, the polymeric matrix is formed witha polyglycolic acid with an average fiber diameter of 15 μm andconfigured into a bladder shaped mold using 4-0 polyglactin 910 sutures.The resulting structure is coated with a liquefied copolymer, such as,for example, pol-DL-lactide-co-glycolide 50:50, 80 milligram permilliliter methylene chloride, in order to achieve adequate mechanicalcharacteristics and to set its shape.

[0034] Polymeric matrixes can be treated with additives or drugs priorto implantation (before or after the polymeric matrix is seeded withcells, if the optional seeded cells are employed), e.g., to promote theformation of new tissue after implantation. Thus, for example, growthfactors, cytokines, extracellular matrix components, and other bioactivematerials can be added to the polymeric matrix to promote graft healingand formation of new tissue. Such additives will in general be selectedaccording to the tissue or organ being reconstructed or augmented, toensure that appropriate new tissue is formed in the engrafted organ ortissue (for examples of such additives for use in promoting bonehealing, see, e.g., Kirker-Head, C.A. Vet. Surg. 24 (5): 408-19 (1995)).For example, when polymeric matrixes (optionally seeded with endothelialcells) are used to augment vascular tissue, vascular endothelial growthfactor (VEGF), (see, e.g., U.S. Pat. No. 5,654,273) can be employed topromote the formation of new vascular tissue. Growth factors and otheradditives (e.g., epidermal growth factor (EGF), heparin-bindingepidermal-like growth factor (HBGF), fibroblast growth factor (FGF),cytokines, genes, proteins, and the like) can be added in amounts inexcess of any amount of such growth factors (if any) which may beproduced by the cells seeded on the polymeric matrix, if added cells areemployed. Such additives are preferably provided in an amount sufficientto promote the formation of new tissue of a type appropriate to thetissue or organ, which is to be repaired or augmented ( e.g., by causingor accelerating infiltration of host cells into the graft). Other usefuladditives include antibacterial agents such as antibiotics.

[0035] One preferred supporting matrix is composed of crossing filamentswhich can allow cell survival by diffusion of nutrients across shortdistances once the cell support matrix is implanted. The cell supportmatrix becomes vascularized in concert with expansion of the cell massfollowing implantation.

[0036] The building of three-dimensional structure constructs in vitro,prior to implantation, facilitates the eventual terminal differentiationof the cells after implantation in vivo, and minimizes the risk of aninflammatory response towards the matrix, thus avoiding graftcontracture and shrinkage.

[0037] The polymeric matrix may be shaped into any number of desirableconfigurations to satisfy any number of overall system, geometry orspace restrictions. For example, in the use of the polymeric matrix forbladder reconstruction, the matrix may be shaped to conform to thedimensions and shapes of the whole or a part of a bladder. Naturally,the polymeric matrix may be shaped in different sizes and shapes toconform to the bladders of differently sized patients. Optionally, thepolymeric matrix should be shaped such that after its biodegradation,the resulting reconstructed bladder may be collapsed when empty in afashion similar to a natural bladder. The polymeric matrix may also beshaped in other fashions to accommodate the special needs of the .patient. For example, a previously injured or disabled patient, may havea different abdominal cavity and may require a bladder reconstructed toadapt to fit. In other embodiments of the invention, the polymericmatrix is used for the treatment of laminar structures in the body suchas urethra, vas deferens, fallopian tubes, lacrimal ducts. In thoseapplications the polymeric matrix may be shaped as a hollow tube.

[0038] The polymeric matrix may be sterilized using any known methodbefore use. The method used depend on the material used in the polymericmatrix. Examples of sterilization methods include steam, dry heat,radiation, gases such as ethylene oxide, gas and boiling.

[0039] Harvesting Cells for the Reconstructive Urothelial Graft (RUG)

[0040] The RUG is constructed in part using urothelial cells and smoothmuscle cells from a donor. One advantage of the methods of the inventionis that because of the rapid growth of the urothelial and smooth musclecells, sufficient cells for the construction of a RUG may be grown inless than 5 weeks. In an autologous RUG, the cells may be derived fromthe patient's own tissue such as, for example, from the bladder,urethra, ureter, and other urogenital tissue. In an allogeneic RUG, thecells may be derived from other member of the patient's species. In axenogenic RUG, the cells may be derived from a species different fromthe patient. Donor cells may be from any urothelial cells and smoothmuscle cells origin and from any mammalian source such as, for example,humans, bovine; porcine, equine, caprine and ovine sources. Urothelialcells and smooth muscle cells may be isolated in biopsies, or autopsies.In addition, the cells may be frozen or expanded before use.

[0041] To prepare for RUG construction, tissue containing urothelial andsmooth muscle cells is dissociated separately into two cell suspensions.Methods for the isolation and culture of cells were discussed in issuedU.S. Pat. number 5,567,612 which is herein specifically incorporated byreference. Dissociation of the cells to the single cell stage is notessential for the initial primary culture because single cell suspensionmay be reached after a period, such as, a week, of in vitro culture.Tissue dissociation may be performed by mechanical and enzymaticdisruption of the extracellular matrix and the intercellular junctionsthat hold the cells together. Urothelial cells and smooth muscle cellsfrom all developmental stages, such as, fetal, neonatal, juvenile toadult may be used.

[0042] Cells (such as autologous cells) can be cultured in vitro, ifdesired, to increase the number of cells available for seeding on thepolymeric matrix “scaffold.” The use of allogenic cells, and morepreferably autologous cells, is preferred to prevent tissue rejection.However, if an immunological response does occur in the subject afterimplantation of the RUG, the subject may be treated withimmunosuppressive agents such as, for example, cyclosporin or FK506, toreduce the likelihood of rejection of the RUG. In certain embodiments,chimeric cells, or cells from a transgenic animal, can be seeded ontothe polymeric matrix.

[0043] Cells may be transfected prior to seeding with genetic material.Useful genetic material may be, for example, genetic sequences which arecapable of reducing or eliminating an immune response in the host. Forexample, the expression of cell surface antigens such as class I andclass II histocompatibility antigens may be suppressed. This may allowthe transplanted cells to have reduced chance of rejection by the host.In addition, transfection could also be used for gene delivery.Urothelial and muscle cells could be transfected with specific genesprior to polymer seeding. The cell-polymer construct could carry geneticinformation required for the long term survival of the host or thetissue engineered neo-organ. For example, cells may be transfected toexpress insulin for the treatment of diabetes.

[0044] Cell cultures may be prepared with or without a cellfractionation step. Cell fractionation may be performed usingtechniques, such as florescent activated cell sorting, which is known tothose of skill in the art. Cell fractionation may be performed based oncell size, DNA content, cell surface antigens, and viability. Forexample, urothelial cells may be enriched and smooth muscle cells andfibroblast cells may be reduced for urothelial cell collection.Similarly, smooth muscle cells may be enriched and urothelial cells andfibroblast cells may be reduced for smooth muscle cell collection. Whilecell fractionation may be used, it is not necessary for the practice ofthe invention.

[0045] Cell fractionation may be desirable, for example, when the donorhas diseases such as bladder cancer or metastasis of other tumors to thebladder. A bladder cell population may be sorted to separate malignantbladder cells or other tumor cells from normal noncancerous bladdercells. The normal noncancerous bladder cells, isolated from one or moresorting techniques, may then be used for bladder reconstruction.

[0046] Another optional procedure in the method is cryopreservation.Cryogenic preservation may be useful, for example, to reduce the needfor multiple invasive surgical procedures. Cells taken from a bladdermay be amplified and a portion of the amplified cells may be used andanother portion may be cryogenically preserved. The ability to amplifyand preserve cells allow considerable flexibility in the choice of donorcells. For example, cells from a histocompatible donor, may be amplifiedand used in more than one recipient.

[0047] Another example of the utility of cryogenic preservation is intissue banks. Donor cells may be cryopreserved along withhistocompatibility data. Donor cells may be stored, for example, in adonor tissue bank. As tissue is needed for bladder reconstruction, cellsmay be selected which are most histocompatible to the patient. Patientswho have a disease or undergoing treatment which may endanger theirbladders may cryogenically preserve a biopsy of their bladders. Later,if the patient's own bladder fails, the cryogenically preserved bladdercells may be thawed and used for treatment. For example, if bladdercancer reappeared after bladder reconstruction, cryogenically preservedcells may be used for bladder reconstruction without the need isolatemore tissue from the patient for culture.

[0048] Seeding

[0049] Seeding of cells onto the polymeric matrix can be performed,e.g., as is described in the Example or according to standard methods.For example, the seeding of cells onto polymeric substrates for use intissue repair has been reported (see, e.g., Atala, A. et al., J. Urol.148 (2 Pt 2): 658-62 (1992); Atala, A., et al. J. Urol. 150 (2 Pt 2):608-12 (1993)). Cells grown in culture can be trypsinized to separatethe cells, and the separated cells can be seeded on the polymericmatrix. Alternatively, cells obtained from cell culture can be liftedfrom a culture plate as a cell layer, and the cell layer can be directlyseeded onto the polymeric matrix without prior separation of the cells.

[0050] In a preferred embodiment, at least 50 million cells aresuspended in media and applied to each square centimeter of a surface ofa polymeric matrix. The polymeric matrix is incubated under standardculturing conditions, such as, for example, 37° 5% CO₂, for a period oftime until the cells attached. However, it will be appreciated that thedensity of cells seeded onto the polymeric substrate can be varied. Forexample, greater cell densities promote greater tissue formation by theseeded cells, while lesser densities may permit relatively greaterformation of tissue by cells infiltrating the graft from the host. Otherseeding techniques may also be used depending on the polymeric matrixand the cells. For example, the cells may be applied to the polymericmatrix by vacuum filtration. Selection of cell types, and seeding ofcells onto a polymeric matrix, will be routine to one of ordinary skillin the art in light of the teachings herein.

[0051] In an embodiment of the invention, a polymeric matrix is seededon two sides with two different populations of cells. This may beperformed by first seeding one side of the polymeric matrix and thenseeding the other side. For example, the polymeric matrix may be placedwith one side on top and seeded. Then the polymeric matrix may berepositioned so that a second side is on top. The second side may thenbe seeded with a second population of cells. Alternatively, both sidesof the polymeric matrix may be seeded at the same time. For example, twocell chambers may be positioned on both sides (i.e., a sandwich) of thepolymeric matrix. The two chambers may be filled with different cellpopulations to seed both sides of the polymeric matrix simultaneously.The sandwiched polymeric matrix may be rotated, or flipped frequently toallow equal attachment opportunity for both cell populations:simultaneous seeding may be preferred when the pores of the polymericmatrix are sufficiently large for cell passage from one side to theother side. Seeding the polymeric matrix on both sides simultaneouslywill reduce the likelihood that the cells would migrate to the oppositeside.

[0052] In another embodiment of the invention, two separate polymericmatrixes may be seeded with different cell populations. After seeding,the two matrixes may be attached together to form a single polymericmatrix with two different cell populations on the two sides. Attachmentof the matrixes to each other may be performed using standard proceduressuch as fibrin glue, liquid co-polymers, sutures and the like.

[0053] Surgical Reconstruction

[0054] Grafting of polymeric matrixes to an organ or tissue to beaugmented can be performed according to the methods described in theExamples or according to art-recognized methods. As shown in theexamples, the polymeric matrix can be grafted to an organ or tissue ofthe subject by suturing the graft material to the target organ.

[0055] The techniques of the invention may also be used to treat cancerof the bladder. For example, a normal bladder tissue sample may beexcised from a patient suffering from bladder cancer. Urothelial cellsand smooth muscle cells from the tissue sample may be cultured for aperiod of time in vitro and expanded. The cells may be sorted using aflorescent activated cell sorter to remove cancerous or precancerouscells. The sorted cells may be used to construct a RUG. At the sametime, the patient may be treated for cancer. Cancer treatment mayinvolve excision of the cancerous part of the bladder in addition tochemotherapy or radiation treatment. After the cancer treatment, the RUGmay be used to reconstruct the bladder.

[0056] While a method for bladder reconstruction is disclosed in theExample, other methods for attaching a graft to an organ or tissue ofthe subject (e.g., by use of surgical staples) may also be employed.Such surgical procedures can be performed by one of ordinary skill inthe art according to known procedures.

[0057] As a result of these benefits, the present method of bladderreconstructive surgery is suitable for bladder tissue repair undernumerous circumstances. As described above the bladder graft may be usedto repair a deteriorated bladder due to.

[0058] Other embodiments and advantages of the invention are set forth,in part, in the description which follows and, in part, will be obviousfrom this description and may be learned from practice of the invention.

EXAMPLES Example 1 Creation of Bladder-Shaped Polymers

[0059] A synthetic polymer matrix of polyglycolic acid with an averagefiber diameter of about 15 μm and an interfiber distance between about 0to about 200 μm and dimensions of about 10 cm by about 10 cm wasconfigured into a bladder shaped mold using biodegradable 4-0polyglactin 910 sutures. The resulting flexible scaffold was coated witha liquefied copolymer, a mixture of about 50%poly-DL-lactide-coglycolide and about 50% 80 mg/ml methylene chloride,in order to achieve adequate mechanical characteristics. Aftersterilization with ethylene oxide, the polymers were stored in adesiccator.

Example 2 Cell Harvest and Culture

[0060] A total of 14 beagle dogs underwent a trigone-sparing cystectomy.The animals were randomly assigned to one of three groups. Two wereassigned to Group A and underwent closure of the trigone without areconstructive procedure. Six were assigned to Group B and underwentbladder reconstruction with a cell-free bladder shaped biodegradablepolymer. Six were assigned to Group C and underwent bladderreconstruction using a prefabricated tissue engineered neo-organ. Theneo-organ comprises a bladder shaped biodegradable polymer withautologous urothelial cells attached to the luminal surface and smoothmuscle cells attached to the exterior surface. The cell populations hadbeen separately expanded from a previously harvested autologoustransmural bladder specimen.

[0061] The six animals in group C, which were to be reconstructed with atissue engineered neo-organ, underwent a transmural bladder biopsy ofabout one square centimeter which was harvested from the vesical domevia a minimal suprapubic midline incision under general anesthesia. Thedefect was closed with a 4-0 polyglactin 910 suture. The bladderspecimens were kept in prewarmed keratinocyte medium, and cell harvestfor in-vitro cultures was initiated immediately after tissue excision.

[0062] Urothelial and smooth muscle cell populations, dissociated fromthe one square centimeter bladder biopsies, could be routinely expandedand passaged separately. The average time elapsed between the initialbladder biopsy and final implantation of the tissue engineeredneo-organs was 32+/−2.8 days (Mean+SD). Approximately thirty-two 25 cmplates of each cell type, muscle and urothelial cells, containingapproximately 10⁷ cells per plate, were processed to constitute onetissue engineered neo-organ.

[0063] The harvested cells were cultured according to previouslypublished protocols of Atala et al., (J. Urol. 150: 608, 1993) andCilento et al., (J. Urol. 152: 655, 1994.) which are herein specificallyincorporated by reference. The urothelial and muscular layers of thebladder biopsy were microsurgically detached from each other andprocessed separately. Briefly, the dissected smooth muscle tissue wascut into cubes of about one millimeter and primarily plated on a 10 cmtissue culture petri dish. Smooth muscle cultures were maintained andexpanded with Dulbeccos's Modified Eagles Medium (DMEM, Sigma, St.Louis, Mo.) supplemented with 10% fetal calf serum (Biowhittaker Inc.,Walkersville, Md.). Urothelial cells were also dissected into onemillimeter cubes and plated on 24 well plates. Urothelial cultures weremaintained and expanded with serum-free keratinocyte growth mediumsupplemented with about 5 ng/ml of epidermal growth factor and about 50μg/ml of bovine pituitary extract (Gibco BRL, Life Technologies, GrandIsland, N.Y.). All cell cultures were incubated at 37° C. in ahumidified atmosphere maintained at about 5% level of carbon dioxide.Medium was changed twice weekly. For cell passage cultures at about 80%confluence were trypsinized by incubation for 5 minutes in 0.05% trypsinin 1 millimole ethylenediaminetetraacetic acid. After this periodsoybean trypsin inhibitor, at 2 units per unit of trypsin, was added tothe cell suspension. Both urothelial and smooth muscle cells wereexpanded separately until sufficient cell quantities were available fora seeding density of approximately one million cells per squarecentimeter of polymer surface.

Example 3 Cell Seeding on Polymer Scaffold

[0064] For each tissue engineered neo-organ, about 32 confluent 25 cmplates of each cell type, muscle and urothelium, were processed forseeding. Muscle cell cultures were trypsinized, collected, washed andcombined in one tube. The exterior surface of the pre-molded bladdershaped polymer matrix was seeded with the resuspended smooth muscle cellpopulation. The cell-seeded polymers were incubated in Dulbeccos'sModified Eagles Medium (DMEM, Sigma, St. Louis, Mo.) supplemented with10% fetal calf serum (Biowhittaker Inc., Walkersville, Md.). The mediumwas changed at 12 hour intervals to ensure sufficient supply ofnutrients. After 48 hours of incubation, the urothelial cells wereprocessed in a similar fashion and were seeded onto the luminal surfaceof the polymer.

Example 4 Bladder Reconstruction

[0065] Following pretreatment with intramuscular injection of 0.1 mg ofacepromazine for every kilogram of body weight, surgery was performedunder intravenous pentobarbital anesthesia of about 25 to about 35 mgper kilogram of body weight with endotracheal aeration: About 500 mg ofCefazolin sodium was administered intravenously both preoperatively andintraoperatively. Additional treatment of subcutaneously Cefazolinsodium was administered for 5 postoperative days at a dose of about 30milligrams per kilogram body weight per day. Postoperative analgesictreatment was managed with subcutaneous injections of about 0.1 to about0.6 milligrams of butorphanol per kilogram of body weight.

[0066] As shown in FIG. 1A, a midline laparotomy was performed, thebladder was exposed (FIG. 1A) and both ureters were identified. Thebladder wall was incised ventrally and both ureteric junctions werevisualized and temporarily intubated with 4 F stents. A subtotalcystectomy was performed, sparing the trigone area bearing the urethraand ureteral junctions. Care was taken not to compromise or obstruct theureters. In two animals the trigone was closed, without any polymergraft, with two layers of 4-0 vicryl. As depicted in FIG. 1B, 12 animalsundergone an anastomosis between the bladder shaped polymer matrix andthe trigone with interlocking running sutures of 4-0 vicryl. Of the 12animals, 6 received a bladder shaped polymer alone, and six received abladder shaped polymer coated with cells. A 10 F silicone catheter wasinserted into the urethra from the trigone in a retrograde fashion. An 8F suprapubic catheter was brought into the bladder lumen passing througha short submucosal tunnel in the trigonal region. The suprapubiccatheter was secured to the bladder serosa with a pursestring suture of4-0 chromic. The anastomosis between trigone and graft was marked ateach quadrant with permanent polypropylene sutures for future graft siteidentification. The neo-bladder was covered with fibrin glue (VitexTechnologies Inc., New York, N.Y.). As depicted in FIG. 1C, omentum waswrapped and secured around the neo-reservoir. The abdomen was closedwith three layers of 3-0 vicryl. After recovery from anesthesia, allanimals wore restraint collars to avoid wound and catheter manipulationduring the early postoperative period. The transurethral catheters wereremoved between postoperative days 4 and 7. Cystograms were performedabout four weeks postoperatively, immediately prior to the suprapubiccatheter removal. Cystograms and urodynamic studies were seriallyperformed at about 1, about 2, about 3, about 4, about 6 and about 11months after surgery.

Example 5 Analysis of Reconstructed Bladder

[0067] Urodynamic studies and radiographic cystograms were performedpreoperatively and postoperatively at about 1, about 2, about 3, about4, about 6, and about 11 months after surgery. The two animals whounderwent closure of the trigone without a reconstructive procedure weresacrificed at about 11 months. Animals from the remaining twoexperimental groups were sacrificed at about 1, about 2, about 3, about4, about 6 and about 11 months after surgery. Bladders were retrievedfor gross, histological and immunocytochemical analyses.

[0068] Urodynamic studies were performed using a 7 F double-lumentransurethral catheter. The bladders were emptied and intravesicalpressures were recorded during instillation of prewarmed saline solutionat constant rates. Recordings were continued until leak point pressures(LPP) were reached. Bladder volume at capacity (Vol_(max)), LLP andbladder compliance (Vol_(max)/LLP) were documented. Bladder compliance ,also called bladder elastance, denotes the quality of yielding topressure or force without disruption. Bladder compliance is also anexpression of the measure of the ability to yield to pressure or forcewithout disruption, as an expression of the distensibility of thebladder. It is usually measured in units of volume change per unit ofpressure change. Subsequently, radiographic cystograms were performed.The bladders were emptied and contrast medium was instilledintravesically under fluoroscopic control. Urodynamic Results Prior totrigone-sparing cystectomy, the animals of groups A, B and C did notsignificantly differ in preoperative mean bladder capacity (78+/−16 ml,63+/−22 ml, 69+/−8 ml, p=0.44, [Means +/−A SD]) or preoperative bladdercompliance (2.6+−0.2 ml/cm H₂O, 2.2+/−1.2 ml/cm H₂O, 2.1+/−1.1 ml/cmH₂O, p=0.85, [Means +/−SD]).

[0069] Both control animals, which did not undergo reconstruction aftersubtotal cystectomy, could only maintain 22% (+/−2%) of the nativecapacity during the observed period. A pattern of frequent voiding wasobvious in these animals. The animals reconstructed with cell-freepolymers developed mean bladder capacities of 46% (+/−20%) ofpreoperative values. A mean bladder capacity of 95% (+/−9%) of theoriginal pre-cystectomy volume was achieved by the tissue engineeredbladder replacements (FIG. 2 A).

[0070] The subtotal cystectomy bladders which were not reconstructedshowed a pronounced reduction in bladder compliance to mean values of10% (+/−3%) of the preoperative values. All polymer only implantswithout cells also had a considerable loss of compliance. At varioustime points of sacrifice, bladder compliances were reduced to an averageof 42% (+/−21%) of the preoperative values. The compliance of the tissueengineered bladders showed almost no difference from the preoperativevalues measured when the native bladder was present (106% +/−16%, FIG. 2B).

[0071] Clinically, all animals had a stable course after bladderreconstruction, were able to void spontaneously upon catheter removaland survived their intended study periods. One month after surgery, theradiographic cystograms showed a watertight reservoir in all animals.Cystograms of the subtotal cystectomy only animals showed thatunaugmented trigones were only able to regenerate minimal reservoircapacities throughout the study period. The polymer only implantsdemonstrated signs of partial graft collapse. The tissue engineeredbladders were fully distendable and their contour could be delineatedfrom the native trigone. During follow-up cystograms, the polymer onlyimplants continued to show smaller sized reservoirs while the tissueengineered bladders appeared normal in size and configuration (FIG. 3).

Example 6 Gross Findings

[0072] At the intended time points, the animals were euthanized byintravenous pentobarbital administration. The internal organs and theurogenital tract were inspected for gross abnormalities. The bladder wasretrieved and the marking sutures identifying the transition zonebetween native trigone and graft were exposed. Cross sections were takenfrom within the native trigone, the outlined transition zone and theproximally located neo-bladder.

[0073] Trigone-Sparing Cystectomy only (Group A ). The reservoirsappeared small, but normal in appearance (FIGS. 4A and B).

[0074] Polymer only Bladders (Group B): Gross inspection of thecell-free polymer implant retrieved at one month showed that theoriginal spherical architecture of the polymer had partially collapsed.A solitary, asymptomatic bladder stone of 11 mm was found in the 2 monthtime point, representing the only incidence of lithogenesis in thisstudy. At the two month time point, graft shrinkage of approximately 50%was macroscopically obvious at necropsy. The bladders retrieved at 4, 6and 11 months contained progressive formations of thick scar tissue atthe dome and were firmly covered with adherent omentum (FIGS. 4 C andD). By 11 months, graft shrinkage of over 90% was obviousmacroscopically. The initially placed polypropylene marking sutures werenoted in the area of the trigone, adjacent to the scar tissue.Approximately 10% of the total bladder area was above the markingsutures.

[0075] Tissue Engineered Neo-Organs (Group C): Autopsy explorationshowed no signs of upper tract obstruction, lithogenesis, encrustrationor other abnormalities for all investigated time points. At one month,the polymer scaffold inside the omentum-wrapped tissue engineeredneo-bladder remained visually and palpably identifiable. Theneo-bladders had a flexible, and distendable configuration. At 6 and 11months, omental adhesions could be bluntly separated from the bladderdome, and a serosa-like layer had regenerated over the tissue engineeredneo-organ (FIG. 4E). The initially placed polypropylene marking sutureswere noted in the distal region of the bladder, at the level of thetrigone. Approximately 70% of the total bladder area was above themarking sutures. Upon entering the bladder ventrally, a smooth mucosalsurface was noted, without any differences between the native and tissueengineered bladder (FIG. 4F).

[0076] During the duration of the study, none of the dogs demonstratedany effects. All animals survived until the time of sacrifice withoutany noticeable complications such as urinary tract infection or calculiformation. Fluoroscopic cystography of all the augmented bladders showeda normal bladder configuration without any leakage at one, two and threemonths after the procedure At retrieval, the augmented bladders appearedgrossly normal without any evidence of diverticular formation in theregion of the graft. The thickness of the grafted segment was similar tothat of the native bladder tissue. There was no evidence of adhesion orfibrosis. Histologically, all retrieved bladders contained a normalcellular organization consisting of a urothelial lined lumen surroundedby submucosal tissue and smooth muscle. An angiogenic response wasevident in all specimens.

Example 7 Histological and Immunocytochemical Findings

[0077] Specimens were fixed in 10% buffered formalin and processed.Tissue sections were cut at about 4 to about 6 microns for routinestaining with Hematoxylin and Eosin (H&E) and Masson's trichrome.Immunocytochemical staining methods were employed with several specificprimary antibodies in order to characterize urothelial and smooth musclecell differentiation in the retrieved bladders. Anti-Desmin antibody(monoclonal NCL-DES-DERII, clone DE-R-11, Novocastra®, Newcastle UK),which reacts with parts of the intermediate filament muscle cell proteindesmin, and Anti-Alpha Smooth Muscle Actin antibody (monoclonal NCL-SMA,clone asm-1, Novocastra®, Newcastle UK), which labels bladder smoothmuscle actin, were used as general markers for smooth muscledifferentiation. Anti-Pancytokeratins AE1/AE3 antibody (monoclonal, Cat.No.1124 161, Boehringer Mannheim®) and AntiCytokeratin 7 antibody(NCL-CK7, Clone LP5K, IgG2 b, Novocastra®, New Castle, UK) which reactagainst intermediate filaments that form part of the cytoskeletalcomplex in epithelial tissues, were used to identify urothelium.Anti-Asymmetric Unit Membrane (AUM) staining, using polyclonalantibodies, was used to investigate the presence of mammalianuroplakins, which form the apical plaques in mammalian urothelium andplay an important functional role during advanced stages of urothelialdifferentiation. Anti S-100 antibody (Sigma®; St. Louis Mo., No.IMMH-9), reacting with the acidic calcium-binding protein S-100, mainlypresent in Schwann cells and glial elements in the nervous system, wasused to identify neural tissues.

[0078] Specimens were fixed in Carnoy's solution and routinely processedfor immunostaining. High temperature antigen unmasking pretreatment withabout 0.1% trypsin was performed using a commercially available kitaccording to the manufacturer's recommendations (Sigma®, St. Louis Mo.,T-8 128). Antigen-specific primary antibodies were applied to thedeparaffinized and hydrated tissue sections. Negative controls weretreated with plain serum instead of the primary antibody. Positivecontrols consisted of normal bladder tissue. After washing withphosphate buffered saline, the tissue sections were incubated with abiotinylated secondary antibody and washed again. A peroxidase reagentwas added and upon substrate addition, the sites of antibody depositionwere visualized by a brown precipitate. Counterstaining was performedwith Gill's hematoxylin.

[0079] Trigone-Sparing Cystectomy only (Group A): The trigone-sparingcystectomy organs showed a normal histological architecture which wasconfirmed by immunocytochemical staining.

[0080] Polymer only Bladders (Group B): The polymers implanted withoutcells were found to undergo a fibrovascular reaction consisting offibroblast deposition and extensive recruitment of inflammatory cells,including macrophages, and ubiquitous signs of angiogenesis at onemonth. Epithelial coverage was evident throughout the entire polymer.The epithelium stained positive for the broadly reactinganti-pancytokeratins AE1/AE3, anti-cytokeratin 7, and the urotheliumspecific antiAUM. Fibrotic tissue deposition was noted at the sites ofpolymer degradation. The 2, 3 and 4 month time points showed extensionof the native submucosal and muscular layer of the trigone onto thefibrotic polymer region at the transition zone. In the 6 and 11 monthspecimens abundant connective tissue formation had replaced the fullydegraded polymer fibers of the proximally located neo-bladder region.Smooth muscle alpha actin positive cells were only scarcely evident inthis region.

[0081] Tissue Engineered Neo-Organs (Group C): The tissue engineeredneoorgan retrieved at one month showed complete luminal coverage withurothelium. The epithelium stained positive for the broadly reactinganti-pancytokeratins AE1/AE3, anti-cytokeratin 7, and the urotheliumspecific anti-AUM. The polymer fibers carried cell formations stainingpositive for a smooth muscle actin. An adequate angiogenic response wasevident. At two months, before the polymers underwent completebiodegradation, the muscle fibers had a spatial alignment, formingvariably sized bundles. By three months, there was complete polymerdegradation and a tri-layered structure was evident in the proximallylocated neo-bladder region, consisting of a morphologically normaluroepithelial lining over a sheath of submucosa, followed by a layercontaining multiform smooth muscle bundles. Six months postoperatively,an ingrowth of neural tissue was present for the first time as evidencedby S-100 staining. Bladders were found to have matured towards a normalhistological and phenotypic structure as evidenced by its staining withhematoxylin and eosin, trichrome, alpha smooth muscle actin, desmin,pancytokeratins AE1/AE3, cytokeratin 7 and AUM antibodies (FIGS. 5 and6). Histologically and immunocytochemically, there were no markeddifferences present between the 6 month and 11 month time pointbladders.

Example 8 Statistical Findings

[0082] Statistical evaluations were performed on the measurements usinga two-tailed Student's t-test with p-values of less than or equal to0.05 considered significant. The cystectomy only controls and polymeronly grafts maintained average capacities of 22% and 46% of preoperativevalues, respectively. An average bladder capacity of 95% of the originalpre-cystectomy volume was achieved in the tissue-engineered bladderreplacements. The subtotal cystectomy reservoirs which were notreconstructed and polymer only reconstructed bladders showed a markeddecrease in bladder compliance (10% and 42%). The compliance of thetissue engineered bladders showed almost no difference from preoperativevalues that were measured when the native bladder was present (106%).Histologically, the polymer only bladders presented a pattern of normalurothelial cells with a thickened fibrotic submucosa and a thin layer ofmuscle fibers. The retrieved tissue engineered bladders showed a normalcellular organization, consisting of a tri-layer of urothelium,submucosa and muscle. Immunocytochemical analyses for desmin, α-actin,cytokeratin 7, pancytokeratins AE1/AE3 and uroplakin III confirmed themuscle and urothelial phenotype. S-100 staining indicated the presenceof neural structures.

[0083] The animals which had undergone the trigone-sparing cystectomyand were closed primarily gained a minimal amount of reservoir volumeover time but did not approach the pre-cystectomy values. The free graftpolymer only bladders had a slight increase in volume and developedfibrotic neo-bladders, which had a well developed urothelial layer, buta markedly deficient muscular architecture, and were associated with areduced compliance curve. The tissue aneroid neo-bladders were able toapproach and surpass the pre-cystectomy bladder capacities. Thecompliance of these bladders approached the pre-cystectomy values ateach time point, including the four week postoperative examination. Theretrieved tissue engineered bladders showed a normal cellularorganization, consisting of a tri-layer of urothelium, submucosa andmuscle. Immunocytochemical analysis with desmin and smooth muscle alphaactin confirmed the muscle phenotype. Pancytokeratins AE1/AE3,cytokeratin 7, and uroplakin III could be demonstrated byimmunohistochemistry, confirming the urothelial phenotype. PositiveS-100 staining suggested, that an ingrowth of neural structures into thetissue engineered bladders is possible. The tissue engineeredneobladders were able to function normally soon after implantation.Structurally and functionally, they were indistinguishable from nativebladders. Our results show, for the first time, that creation of atri-layered structure, composed of bladder muscle and urothelium invitro, is beneficial for the ultimate functional results of bladdertissue created de-novo. Our results of bladder replacement with thecell-free polymer graft are consistent with prior reports in theliterature over the last several decades regarding free grafts. Whenother materials are used as free grafts without cells, the differenthistological components may be present, but are not necessarily fullydeveloped or functional. Furthermore, the results of the cell-freepolymer bladder control group are consistent with the literature interms of graft contracture and shrinkage over time. The second controlgroup, which underwent primary closure after cystectomy, clearlyindicated that the increase in capacity in the tissue engineeredneo-bladders was due mostly to the implant and not to the naturalregenerating and elastic features of the native canine bladders. Theresults show that bladder submucosa seeded with urothelial and musclecells can form new bladder tissue which is histologically andfunctionally indistinguishable from the native bladder. This resultmaybe due to a possible maintenance of the architectural form of thebladder by the extracellular matrix regenerated by the seeded cells. Theurothelial and muscle cells seeded on the polymeric matrix appear toprevent the resorption of the graft. This technology is able to form newbladder tissue which is anatomically and functionally similar to that ofnormal bladders.

[0084] Other embodiments and uses of the invention will be apparent tothose skilled in the art from consideration of the specification andpractice of the invention disclosed herein. All U.S. Patents and otherreferences noted herein for whatever reason are specificallyincorporated by reference. The specification and examples should beconsidered exemplary only with the true scope and spirit of theinvention indicated by the following claims.

We claim:
 1. A method for the reconstruction, repair, augmentation orreplacement of laminarily organized luminal organs or tissue structuresin a patient in need of such treatment comprising the steps of: a)providing a biocompatible synthetic or natural polymeric matrix shapedto conform to at least a part of the luminal organ or tissue structurein need of said treatment; b) depositing a first cell population on orin a first area of said polymeric matrix; c) depositing a second cellpopulation of a different cell type than said first cell population in asecond area of said polymeric matrix, said second area beingsubstantially separated from said first area; and d) implanting theshaped polymeric matrix cell construct into said patient at the site ofsaid treatment for the formation of laminarily organized luminal organor tissue structure.
 2. The method of claim 1 wherein the biocompatiblematerial is biodegradable.
 3. The method of claim 1 wherein thebiocompatible polymeric matrix is formed from a material selected fromthe group of materials consisting of cellulose ether, cellulose,cellulosic ester, fluorinated polyethylene, phenolic,poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,polyester, polyestercarbonate, polyether, polyetheretherketone,polyetherimide, polyetherketone, polyethersulfone, polyethylene,polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenyleneoxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythiether, polytriazole,polyurethane, polyvinyl, polyvinlidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, or copolymers or physical blends thereof.4. The method of claim 1 wherein the biocompatible material ispolyglycolic acid.
 5. The method of claim 1 wherein the polymeric matrixis comprised of fibers with an interfiber distance between about 0 to1000 μm.
 6. The method of claim 1 wherein the polymeric matrix iscomprised of fibers with an interfiber distance between about 0 to 500μm.
 7. The method of claim 1 wherein the polymeric matrix is comprisedof fibers with an interfiber distance between about 0 to 200 μm.
 8. Themethod of claim 1 wherein the polymeric matrix is coated with abiocompatible and biodegradable shaped setting material.
 9. The methodof claim 8 wherein the shape settling material comprise a liquidcopolymer.
 10. The method of claim 9 wherein the co-polymer comprisespoly-DL-lactide-coglycolide.
 11. The method of claim 1 wherein the firstcell population is substantially a urothelial cell population.
 12. Themethod of claim 1 wherein the second cell population is substantially asmooth muscle cell population.
 13. The method of claim 1 wherein theluminal organ or tissue structure is of genitourinary organ.
 14. Themethod of claim 13 wherein the luminal organ or tissue structure isselected from the group consisting of bladder, ureters and urethra. 15.The method of claim 14 wherein the luminal organ or tissue structure isa bladder or bladder segment and having urothelial cells deposited onthe inner surface of said matrix and smooth muscle cells deposited onthe outer surface of said matrix.
 16. The method of claim 15 wherein thelaminarily organized luminal organ or tissue structure formed in vivoexhibits the compliance of natural bladder tissue.
 17. The method ofclaim 1 wherein said first and second cell populations are depositedsequentially.
 18. The method of claim 1 wherein said first and secondcell populations are deposited on separate matrix layers and said matrixlayers are combined after the deposition steps.
 19. A device for thereconstruction, repair, augmentation or replacement of laminarilyorganized luminal organs or tissue structures comprising: animplantable, biocompatible, synthetic or natural polymeric matrix havingat least two separate surfaces and shaped to conform to a at least apart of the luminal organ or tissue structure in need of said treatment,and at least two different cell populations deposited in substantiallyseparate areas on or in said polymeric matrix to form a laminarilyorganized matrix/cell construct.
 20. The device of claim 19 wherein thebiocompatible material is biodegradable.
 21. The device of claim 19wherein the biocompatible polymeric matrix is formed from a materialselected from the group of materials consisting of cellulose ether,cellulose, cellulosic ester, fluorinated polyethylene, phenolic,poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,polyester, polyestercarbonate, polyether, polyetheretherketone,polyetherimide, polyetherketone, polyethersulfone, polyethylene,polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenyleneoxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, or copolymers or physical blends thereof.22. The device of claim 19 wherein the biocompatible material ispolyglycolic acid.
 23. The device of claim 19 wherein the polymericmatrix is comprised of fibers with an interfiber distance between about0 to 1000 μm.
 24. The device of claim 19 wherein the polymeric matrix iscomprised of fibers with an interfiber distance between about 0 to 500μm.
 25. The device of claim 19 wherein the polymeric matrix is comprisedof fibers with an interfiber distance between about 0 to 200 μm.
 26. Thedevice of claim 19 wherein the polymeric matrix is coated with abiocompatible and biodegradable shaped setting material.
 27. The deviceof claim 26 herein the shape settling material comprise a liquid .copolymer.
 28. The device of claim 27 wherein the co-polymer comprisespoly-DL-lactide-coglycolide.
 29. The. device of claim 19 wherein thefirst cell population is substantially a urothelial cell population. 30.The device of claim 19 wherein the second cell population issubstantially a smooth muscle cell population.
 31. The device of claim19 wherein the luminal organ or tissue structure is of genitourinaryorgan.
 32. The device of claim 31 wherein the luminal organ or tissuestructure is selected from the group consisting of bladder, ureters andurethra.
 33. The device of claim 32 wherein the luminal organ or tissuestructure is a bladder or bladder segment and having urothelial cellsdeposited on the inner surface of said matrix and smooth muscle cellsdeposited on the outer surface of said matrix.
 34. The device of claim33 wherein the laminarily organized luminal organ or tissue structureformed in vivo exhibits the compliance of natural bladder tissue. 35.The device of claim 19 wherein said first and second cell populationsare deposited sequentially.
 36. The device of claim 19 wherein saidfirst and second cell populations are deposited on separate matrixlayers and said matrix layers are combined after the deposition steps.37. A device for the repair, reconstruction, augmentation or replacementof damaged or missing bladder tissue in a patient in need of suchtreatment comprising: an implantable, biocompatible synthetic or naturalpolymeric matrix shaped to conform to the part of said bladder tissue inneed of said treatment and having urothelial cells deposited on and nearthe inside surface of said matrix, and having smooth muscle cellsdeposited on and near the outside surface of said matrix, wherein uponimplantation into said patient, said device forms a laminarily organizedluminal tissue structure having the compliance of normal bladder tissue.38. The device of claim 37 wherein said shaped matrix is a fibrous meshof a polymer selected from the group consisting of polyglycolic acid,polylactic acid and copolymers or blends thereof; coated with a shaperetaining material.
 39. The device of claim 37 wherein said shaperetaining material is a solution of a hardenable polymer.
 40. The deviceof claim 39 wherein said hardenable polymer is polyDL-lactide-coglycolide.